摘要
目的观察枸杞多糖(lycium barbarum polysaccharide,LBP)对过氧化氢(hydrogen peroxide,H_(2)O_(2))刺激所致的肾小管上皮细胞(HK2)的损伤作用以及磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)/蛋白激酶(protein kinase,AKT)通路蛋白表达的情况。方法孵育HK2细胞,采用H_(2)O_(2)刺激制作损伤模型。根据实验设计分为正常组、LBP对照组、模型组和LBP干预组。正常组:正常孵育;LBP对照组:正常孵育液中滴加LBP;模型组:正常孵育液中滴加H_(2)O_(2);LBP干预组:预先用LBP孵育24 h后再滴加H_(2)O_(2)。H_(2)O_(2)和LBP在孵育液中的含量分别为100μmol/L、100 mg/L。每组均干预24 h。光镜观察细胞形态变化,试剂盒测定其存活率,流式技术仪检验其凋亡率,比色法比较胞质内丙二醛(malondialdehyde,MDA)水平以及超氧化物歧化酶(superoxide dismutase,SOD)活力,蛋白印迹评估胞质内B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl 2-Associated X,Bax)、PI3K、AKT以及磷酸化的PI3K(p-PI3K)、AKT(p-AKT)水平。结果正常组和LBP对照组相比,在形态状况、存活率及凋亡率、胞质内MDA、SOD、Bcl-2、Bax、PI3K、p-PI3K、AKT、p-AKT方面差异均无统计学意义(均P>0.05);模型组与正常组相比,细胞变圆、脱落,存活率[(50.23±3.38)%]降低,凋亡率[(52.49±3.56)%]增加,MDA含量[(4.63±0.69)mmol/L]上升,SOD活力[(9.56±0.71)U/L]下降,胞质内Bcl-2(0.165±0.023)、p-PI3K/PI3K(0.413±0.057)及p-AKT/AKT(0.647±0.063)含量降低,Bax含量(1.058±0.034)升高(均P<0.05);LBP干预组与模型组比较,细胞伸展、脱落少,存活率[(85.31±3.49)%]增加,凋亡率[(24.62±2.97)%]下降,MDA含量[(2.08±0.35)mmol/L]降低,SOD活力[(17.03±1.45)U/L]升高,胞质内Bcl-2(0.854±0.044)、p-PI3K/PI3K(0.718±0.079)及p-AKT/AKT(0.975±0.112)含量增加,Bax含量(0.213±0.023)减少(均P<0.05)。结论LBP能保护H_(2)O_(2)刺激所致的肾小管上皮细胞氧化损伤,增加其存活率并降低凋亡率,减少胞内有害物质,这些均和PI3K/AKT通路的激活紧密相关。
Objective To explore the effect oflycium barbarum polysaccharide(LBP)on the injury of renal tubular epithelial cells(HK2)stimulated by hydrogen peroxide(H_(2)O_(2))and the expression of phosphatidylinositol 3 kinase(PI3K)/protein kinase(AKT)pathway protein.Methods HK2 cells were incubated and the injury model was made by HO2 stimulation.According to the experimental design,it was divided into normal group,LBP control group,model group and LBP intervention group,and four groups were given intervention reagents respectively.Normal group:normal incubation;LBP control group:LBP was added dropwise to the normal incubation solution;Model group:H_(2)O_(2)was added dropwise to the normal incubation solution;LBP intervention group:incubate with LBP for 24 hours in advance,and then add H_(2)O_(2)dropwise.The contents of H_(2)O_(2)and LBP in the incubation solution were 100μmol/L and 100 mg/L respectively.Each group was intervened for 24 hours.The morphological changes of cellswere showed though light microscope,the survival rate was determined by the kit,and the apoptosis ratio was analyzed though flow cytometry.The activity of malondialdehyde(MDA)and superoxide dismutase(SOD)in cytoplasm weremeasured by colorimetry.The levels of B-cell lymphoma-2(Bcl-2),Bcl 2-Associated X(Bax),PI3K,AKT and phosphorylated PI3K(p-PI3K)and AKT(p-AKT)were evaluated by Western blotting.Results Normal group and LBP control group had not differences in cell morphological status,survival and apoptosis rate,intracellular MDA,SOD,Bcl-2,Bax,PI3K,p-PI3K,AKT and p-AKT(all P>0.05).Compared with the normal group,the cells in the model groupbecame round and shed,survival rate[(50.23±3.38)%]decreased,apoptosis rate[(52.49±3.56)%]increased,the content of MDA[(4.63±0.69)mmol/L]increased while the activity of SOD[(9.56±0.71)U/L]decreased,the contents of Bcl-2(0.165±0.023),p-PI3K/PI3K(0.413±0.057)and p-AKT/AKT(0.647±0.063)decreased,and the content of Bax(1.058+0.034)increased(all P<0.05).Compared with the model group,the cells in the LBP intervention group showed extension and less abscission,survival rate[(85.31±3.49)%]increased,apoptosis rate[(24.62±2.97)%]decreased,the content of MDA[(2.08±0.35)mmol/L]decreased while the activity of SOD[(17.03±1.45)U/L]increased,the contents of Bcl-2(0.854±0.044),p-PI3K/PI3K(0.718±0.079)and p-AKT/AKT(0.975±0.112)increased,and the content of Bax(0.213±0.023)decreased(all P<0.05).Conclusion Lycium barbarum polysaccharide can protect renal tubular epithelial cells from oxidative damage induced by H,O2,increase their survival rate,reduce apoptosis rate and reduce intracellular harmful substances,which are closely related to the activation of PI3K/AKT pathway.
作者
宋倩
单铁英
侯鑫
陈楠
单铁强
刘晓丽
SONG Qian;SHAN Tie-ying;HOU Xin;CHEN Nan;SHAN Tie-qiang;LIU Xiao-li(Department of Medicine,First Hospital of Handan,Handan Hebei,056002,China;Medical College,Hebei University of Engineering,Handan Hebei,056002,China;Department of Laboratory,Qinhuangdao Haigang Hospital,Qinhuangdao Hebei,066000,China;Department of Nephrology,Affliated Hospital of Hebei Engineering University,Handan Hebei,056002,China)
出处
《职业与健康》
CAS
2023年第6期764-768,共5页
Occupation and Health
关键词
枸杞多糖
过氧化氢
肾小管上皮细胞
PI3K/AKT通路
氧化损伤
Lycium barbarum polysaccharide
Hydrogen peroxide
Renal tubular epithelial cells
PI3K/AKTpathway
Oxidative damage
